GridTape For Fast Nanoscale Imaging

ABSTRACT

A tape for collecting tissue samples in a manner compatible with imaging in a transmission electron microscopy (TEM) system, includes a tape substrate having two side walls forming a trough. The trough is defined by a bottom surface of the tape substrate and internal surfaces of the side walls, with an open side therebetween. A support film is attached to a top surface of the tape substrate, and a plurality of apertures is spaced at predetermined locations along the length of the tape substrate, each aperture being covered by the support film. The tape includes a stacked configuration in which the tape substrate is wound in layers, the bottom surfaces of the side walls in one layer being in contact with the support film in an immediately adjacent layer. The apertures in the second layer are aligned within the trough of the first layer, between the side walls.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application Ser. No. 62/325,747, filed on Apr. 21, 2016, which ishereby incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.T32MH20017, grant no. T32HL007901, and grant no. R2INS085320, each grantawarded by the National institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to transmission electronmicroscopy (“TEM”), and, more particularly, to a TEM-compatiblecontinuous substrate with a protective configuration.

BACKGROUND OF THE INVENTION

TEM offers higher resolution and faster acquisition rates compared withscanning methods. However, these benefits are typically offset bylaborious techniques for manually collecting each sample onto a separateimaging substrate, which has been required for maintaining the integrityof a nano-scale electron-transparent substrate on which samples rest. Analternative solution to such laborious manual techniques is covering atape-based substrate to produce a tape sandwich, such as disclosed inU.S. Pat. No. 8,366,857, titled “Methods, Apparatus And Systems ForProduction, Collection, Handling, And Imaging Of Tissue Sections.”However, this solution is error-prone and inefficient. The presentdisclosure is directed to solving the above and other needs, including,for example, providing a substrate that allows the use of automatedsample collection and handling techniques.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a tape is directed tocollecting tissue samples in a manner compatible with imaging in TEM.The tape includes a tape substrate having two side walls extending froma bottom surface to form a trough, the trough being defined by thebottom surface of the tape substrate and internal surfaces of the sidewalls. The trough has an open side between bottom surfaces of the sidewalls. A plurality of apertures is spaced at predetermined locationsalong the length of the tape substrate, and an electron-lucent supportfilm is attached to a top surface of the tape substrate. Each of theplurality of apertures is covered by the support film for receiving arespective sample. The tape includes a stacked configuration in whichthe tape substrate is wound in a plurality of layers along its length,the bottom surfaces of the side walls in a first layer of the pluralityof layers being in contact with the support film in an immediatelyadjacent second layer of the plurality of layers. Apertures of theplurality of apertures in the second layer are aligned within the troughof the first layer, between the side walls, for protecting structuralintegrity of the support film over each respective aperture.

According to another aspect of the present invention, a tape is directedto collecting tissue samples in a manner compatible with imaging in aTEM system, and includes a substrate having a cross-section shape in theform of a channel with a top surface, a bottom surface, and a pair ofside walls extending from the bottom surface. The side walls form aninternal space with an open side in proximity with bottom surfaces ofthe side walls. The substrate has a stacked configuration in which thesubstrate is arranged in several concentric layers along its length, theconcentric layers including at least a first layer and an immediatelyadjacent second layer. A plurality of apertures is centrally positionedacross the width of the substrate and equally-spaced along the length ofthe substrate. Each aperture of the plurality of apertures is covered atleast in part by the electron-lucent support film for receiving arespective tissue sample, the support film providing structuralintegrity for receiving a tissue sample over each aperture. The bottomsurfaces of the side walls in the first layer are in contact with thesupport film or the top surface of the substrate in the second layer,each aperture in the second layer being aligned with the open sidebetween the side walls.

Additional aspects of the invention will be apparent to those ofordinary skill in the art in view of the detailed description of variousembodiments, which is made with reference to the drawings, a briefdescription of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an isometric view illustrating a tape for collecting tissuesamples in a manner compatible with imaging in a TEM system.

FIG. 2 is a side view illustrating a TEM system in which tissue samplescollected onto a tape are imaged via a microscope.

FIG. 3A is a cross-sectional view along lines 3A-3A of FIG. 2 andillustrates the tape in an unstacked configuration.

FIG. 3B is a cross-sectional view along lines 3B-3B of FIG. 2 andillustrates the tape in a stacked configuration.

FIG. 4 is a top view illustrating a top surface of the tape of FIG. 1.

FIG. 5 is a bottom view illustrating a trough of the tape of FIG. 1.

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the invention is not intended to belimited to the particular forms disclosed. Rather, the invention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings and will herein he described indetail preferred embodiments of the invention with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit the broadaspect of the invention to the embodiments illustrated. For purposes ofthe present detailed description, the singular includes the plural andvice versa (unless specifically disclaimed); the words “and” and “or”shall be both conjunctive and disjunctive; the word “all” means “any andall”; the word “any” means “any and all”; and the word “including” means“including without limitation.”

Referring to FIG. 1, a tape 100 includes a TEM-compatible continuoustape substrate 102 that allows the use of automated sample collectionand handling techniques. The tape 100 provides direct benefits, forexample, in the collection of large TEM datasets that are common inneuroscience research, which seeks to understand the function of thebrain. In other examples, the tape 100 is beneficial in any field thatuses TEM, including medical diagnostics. Thus, the tape 100 iscompatible with existing automated sample sectioning and collectionapproaches, but has key features that make it possible, for example, touse a transmission electron microscope instead of scanning electronmicroscope systems that are currently used for imaging in theneuroscience field.

The tape 100 includes a plurality of apertures 104, which resembletraditional TEM slot grids, and a support film 106 that is attached to atop surface 108 of the tape substrate to cover each aperture 104.Consequently, the support film provides structural support for tissuesamples 110 deposited unto the tape 100 for TEM imaging. The tape 100further includes a plurality of barcodes 112, each barcode 112 beingaligned with a respective aperture 104 for identifying or referencing arespective tissue sample 110. The tape 100 also includes a plurality ofvents 114, each vent 114 being aligned with a respective aperture forproviding an air and moisture removal path when specific vacuumconditions are desired.

The tape 100 is flexible such that it can achieve a stackedconfiguration 120 in which the tape 100 is rolled unto itself,optionally being spooled unto a supporting reel, resulting in aplurality of concentric layers 122. The tape 100 further has anunstacked configuration 122 in which the tape 100 is unwound in astraight path for observing one or more of the tissue samples 108.

To achieve the stacked and unstacked configuration, the tape 100includes a flexible material such as a polyimide film or other polymers.By way of example, the material includes an aluminum-coated polyimidefilm, a platinum-coated polyimide film, a conductive polyimide film, apolyether ether ketone (PEEK) polymer, and a polyethylene terephthalate(PET) polymer. In another more specific example, the material is aKAPTON® polyimide film. One considering factor for selecting thematerial includes the material's ability to be etched with ultravioletlight. Another factor for selecting the material includes conductivityfor being compatible with imaging requirements.

In accordance with one example, the tape 100 has a thickness TT (along aZ axis) of approximately 125 micrometers, a tape width TW (along a Yaxis) of approximately 8 millimeters, and a tape length TL (along an Xaxis) of up to approximately 30 meters. In accordance with otherexamples, the tape thickness TT is 50 micrometers or larger to providesufficient structural integrity.

Optionally, the tape 100 further includes a plurality of smallinter-slot holes 150 located between apertures 104. According to oneexample, the holes have a generally square shape of approximately 250micrometers×250 micrometers. The inter-slot holes 150 provide abeneficial features in which background images of just the support film106 (on top of the tape 100) are captured. The background images are,then, subtracted from the raw data.

Referring to FIG. 2, a TEM system 130 includes a sending support reel132 on which the tape 100 is initially stored and a receiving supportreel 134 on which the tape 100 is subsequently transferred after tissuesamples 108 have been imaged or observed via a transmission electronmicroscope 136. By way of example, the imaging optionally forms areconstructed three-dimensional image volume 138 associated with thetissue samples 108, and which includes a representative single section139 of the three-dimensional image volume 138. More specifically, in theillustrated example, an electron beam 137 is produced by thetransmission electron microscope 136 to image physical serial sections108 that can be virtually reconstructed into the image volume 138 of alarval zebrafish. The serial sections 108 had been previously cut intothousands of thin sections that were, then, collected onto the tape 100.

Referring to FIG. 3A, a cross-sectional view of an unstacked sectionshows that the tape 100 has a trough 140 formed by two side walls 142,144 extending from a bottom surface 146 of the tape substrate 102. Onebenefit of the trough 140 is that it allows the tape 100 to roll ontoitself, into the stacked configuration 120, without damaging the tissuesamples 108 or the support film 106.

The trough 140 is defined by the bottom surface 146 of the tapesubstrate and internal surfaces 142 a, 144 a of the two side walls 142,144, with an open side 148 extending between bottom surface 142 b, 144 bof the side walls 142, 144. The side walls 142, 144 have externalsurfaces 142 c, 144 c that define outer edges of the tape substrate 102in a transverse direction (i.e., across its width, along the Y-axis).

In the illustrated example, the trough 140 is generally in the form of aC-shaped channel. However, in other embodiments, the trough 140 hasother forms or shapes. By way of a specific example, the trough 140 hasa depth XD of approximately 25 micrometers. In other specific examples,the trough depth XD has a range of approximately 6 micrometers toapproximately 25 micrometers.

Referring to FIG. 3B, the stacked configuration 120 shows a first layer122 a that is immediately adjacent and in contact with a second layer122 b, which is immediately adjacent and in contact with a third layer122 c. Specifically, bottom surfaces 142 b, 144 b of the first layer 122a are in contact with the support film 106 of the second layer 122 b,and bottom surfaces 142 b, 144 b of the second layer are in contact withthe support film 106 of the third layer 122 c. Beneficially, the sidewalls 142, 144 surround the aperture 104 area to remain clear of therespective support film 106. More specifically, a distance D between theinternal surfaces 142 a, 144 a of the side walls 142, 144 is greaterthan an aperture width AW or a sample width SW to prevent physicalcontact with the tissue sample 110. The distance D is less than the tapewidth TW, in accordance with a wall width between respective externalsurfaces 142 c, 144 c and internal surfaces 142 a, 144 a of the sidewall 142, 144. Based on the geometric configuration of the tape 100, thetrough 140 creates a natural protective zone in which the structuralintegrity of a respective tissue sample 110, support film 106, andaperture 104 is maintained without requiring additional, costly films orprotective elements.

Referring to FIG. 4, the barcodes 112 are located in proximity to anedge of the tape 100, have a generally rectangular shape, and aregenerally centered along the length of the tape 100 (along the X-axis)with respect to an associated aperture 104. According to one example, abarcode 112 has a length BL of approximately 5 millimeters and a widthBW of approximately 1 millimeter.

Referring to FIG. 5, the vents 114 are generally centered along thelength of the tape 100 (along the X-axis) with respect to an associatedaperture 104. According to one example, a vent 114 has a gap VG ofapproximately 0.1 micrometers (along the X-axis) and extends the fulldistance of each side wall 142, 144, between respective externalsurfaces 142 c, 144 c, and internal surfaces 142 a, 144 a.

In accordance with a specific example, the apertures 104 have anaperture length AL of approximately 2 millimeters, an aperture width ofapproximately 1.5 millimeters, and rounded corners RC with a radius ofapproximately 0.5 millimeters. In accordance with another specificexample, the apertures 104 have an aperture length AL of approximately 3millimeters, an aperture width of approximately 2 millimeters, androunded corners RC with a radius of approximately 1 millimeter. Inaccordance with yet another specific example, the apertures 104 arespaced apart at a distance AS that is approximately 4 millimeters.

In accordance with one manufacturing method, the tape 100 is made usinga computer numerical control (CNC) ultraviolet (UV) laser system to etchthe protective trough 140 and to cleanly cut the apertures 104 (orslots) into a plastic web material. The CNC UV laser approach providesflexibility for addition of other features into and/or through the tapesubstrate 102, such as the barcodes 112 for section identification, thevents 114 for reducing vacuum pump-down times when imaging tissuesamples 110, and/or calibration marks to help configure the tape 100 forhandling machines.

The tape 100 provides many benefits over traditional TEM substrates. Byway of example, the tape 100 maintains section order, avoids skipping ofevery other aperture position, and allows for automated samplecollection using a commercially available instrument (e.g., ATUMtome,Boeckeler Instruments, Inc.). In another example, the tape 100 isbeneficial because it is compatible with reel-to-reel processing andimaging techniques that greatly improve acquisition rates and reducehandling errors. In yet another example, the tape 100 and method ofmanufacturing is beneficial because the geometry configuration iscustomizable in accordance with specific needs.

Each of these embodiments and obvious variations thereof is contemplatedas falling within the spirit and scope of the claimed invention, whichis set forth in the following claims. Moreover, the present conceptsexpressly include any and all combinations and subcombinations of thepreceding elements and aspects.

1-23. (canceled)
 24. A tape for collecting tissue samples, the tape comprising: a tape substrate having two side walls extending from a bottom surface to form a trough, the tape substrate being windable in a plurality of layers along its length to form a stacked configuration, the plurality of layers including at least a first layer and a second layer; a support film attached to the tape substrate; and a plurality of apertures positioned along the length of the tape substrate and covered by the support film, at least one aperture of the plurality of apertures in the second layer being aligned within the trough of the first layer.
 25. The tape of claim 24, wherein the trough is defined by the bottom surface of the tape substrate and internal surfaces of the side walls, the trough having an open side between bottom surfaces of the side walls.
 26. The tape of claim 24, wherein each aperture of the plurality of apertures is equally spaced from each adjacent aperture of the plurality of apertures.
 27. The tape of claim 24, further comprising a plurality of vents in each of the side walls.
 28. The tape of claim 24, further comprising a plurality of barcodes along the top surface of the tape substrate.
 29. The tape of claim 24, wherein the tape substrate is selected from a group consisting of aluminum-coated polyimide film, platinum-coated polyimide film, polyimide film, conductive polyimide film, polyether ether ketone (PEEK) polymer, and polyethylene terephthalate (PET) polymer.
 30. The tape of claim 24, wherein the support film is selected from a group consisting of polyimide film and electron-lucent film.
 31. The tape of claim 24, further comprising a plurality of inter-slot holes positioned between adjacent ones of the plurality of apertures.
 32. A tape for collecting tissue samples, the tape comprising: a substrate having a stacked configuration in which the substrate is arranged in several concentric layers along its length, the concentric layers including at least a first layer and an immediately adjacent second layer, the substrate having a top surface, a bottom surface, and side walls extending from the bottom surface; a plurality of apertures spaced along the length of the substrate; and a support film covering at least in part each aperture of the plurality of apertures, the support film being in contact with the bottom surface of the first layer.
 33. The tape of claim 32, further comprising a plurality of vents in each of the side walls.
 34. The tape of claim 32, further comprising a plurality of barcodes along the top surface of the substrate.
 35. The tape of claim 32, wherein the substrate is selected from a group consisting of aluminum-coated polyimide film, platinum-coated polyimide film, polyimide film, conductive polyimide film, polyether ether ketone (PEEK) polymer, and polyethylene terephthalate (PET) polymer.
 36. The tape of claim 32, wherein the support film is selected from a group consisting of polyimide film and electron-lucent film.
 37. The tape of claim 32, further comprising a plurality of inter-slot holes positioned between adjacent ones of the plurality of apertures.
 38. A method for collecting tissue samples on a tape, the method comprising: stacking a tape substrate in several concentric layers along its length, the concentric layers including at least a first layer and an immediately adjacent second layer, the tape substrate having a trough; and receiving a respective tissue sample in one or more apertures of a plurality of apertures, the plurality of apertures being spaced along the length of the tape substrate and being covered by a support film, at least one aperture of the plurality of apertures in the second layer being aligned within the trough of the first layer.
 39. The method of claim 38, further comprising initially storing the tape on a sending support reel.
 40. The method of claim 39, further comprising subsequently transferring the tape on a receiving support reel after collected tissue samples are imaged or observed.
 41. The method of claim 39, further comprising imaging or observing the collected tissue samples via a transmission electron microscope.
 42. The method of claim 41, further comprising forming a reconstructed three-dimensional image volume that is associated with the collected tissue samples.
 43. The method of claim 42 wherein the reconstructed three-dimensional image volume includes a representative single section. 